The intracellular tyrosine kinase, Ack1 is a ˜141 kDa protein with amino terminal sterile alpha motif (SAM) domain, kinase domain, Src homology 3 (SH3) domain, Cdc42/Rac interactive-binding (CRIB) domain, proline rich domain and UBA domain at the carboxy terminus (FIG. 22) (Manser, E., Leung, T., Salihuddin, H., Tan, L. & Lim, L. A non-receptor tyrosine kinase that inhibits the GTPase activity of p21cdc42. Nature 363, 364-367 (1993); Galisteo, M. L., Yang, Y., Urena, J. & Schlessinger, J. Activation of the nonreceptor protein tyrosine kinase Ack occurs by multiple extracellular stimuli. Proc Natl Acad Sci USA 103, 9796-9801 (2006)). AKT plays a central role in growth, proliferation and cell survival (Manning B D, Cantley L C (2007) AKT/PKB signaling: navigating downstream. Cell 129: 1261-1274; Bellacosa A, et al. (2005) Activation of AKT kinases in cancer: implications for therapeutic targeting. Adv Cancer Res 94: 29-86; Vivanco I, Sawyers C L (2002) The phosphatidylinositol 3-Kinase AKT pathway in human cancer. Nat Rev Cancer 2: 489-501). AKT activation occurs when ligand binding to RTK facilitates translocation of AKT to the plasma membrane (Franke T F, et al. (1995). The protein kinase encoded by the Akt proto-oncogene is a target of the PDGF-activated phosphatidylinositol 3-kinase. Cell 81: 727-736; Burgering B M, Coffer P J (1995) Protein kinase B (c-Akt) in phosphatidylinositol-3-OH kinase signal transduction. Nature 376: 599-602; Stephens L, et al. (1998) Protein kinase B kinases that mediate phosphatidylinositol 3,4,5-trisphosphate-dependent activation of protein kinase B. Science 279: 710-714; Stokoe D, et al. (1997) Dual role of phosphatidylinositol-3,4,5-trisphosphate in the activation of protein kinase B. Science 277: 567-570) where it is phosphorylated at Thr308 by phosphoinositide-dependent protein kinase-1 (PDK1) and at Ser473 by the ‘PDK2’, a class of about 10 different kinases (Dong L Q, Liu F (2005) PDK2: the missing piece in the receptor tyrosine kinase signaling pathway puzzle. Am J Physiol Endocrinol Metab 289: E187-196) including the mTORC2 complex (Sarbassov D D, et al (2005) Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex. Science 307: 1098-1101). Although RTKs do not directly phosphorylate Ack1, they facilitate Ack1 autophosphorylation in a ligand dependent manner (Mahajan, N. P., Whang, Y. E., Mohler, J. L. & Earp, H. S. Activated tyrosine kinase Ack1 promotes prostate tumorigenesis: role of Ack1 in polyubiquitination of tumor suppressor Wwox. Cancer Res 65, 10514-10523 (2005)). Phosphorylation of AKT at Thr308 and Ser473 leads to its kinase activation (Alessi D R, et al. (1996) Mechanism of activation of protein kinase B by insulin and IGF-1. Embo J 15: 6541-6551). Upon activation, AKT phosphorylates its substrates to transduce survival signals (Manning B D, Cantley L C (2007) AKT/PKB signaling: navigating downstream. Cell 129: 1261-1274; Vivanco I, Sawyers C L (2002) The phosphatidylinositol 3-Kinase AKT pathway in human cancer. Nat Rev Cancer 2: 489-501; Greer E L, Brunet A (2005) FOXO transcription factors at the interface between longevity and tumor suppression. Oncogene 24: 7410-7425; Huang H, Tindall D J (2007) Dynamic FoxO transcription factors. J Cell Sci 120: 2479-2487).
Activation of protein kinase AKT/PKB is common occurrence in variety of human cancers (Manning, B. D. & Cantley, L. C. AKT/PKB signaling: navigating downstream. Cell 129, 1261-1274 (2007); Bellacosa, A., Kumar, C. C., Di Cristofano, A. & Testa, J. R. Activation of AKT kinases in cancer: implications for therapeutic targeting. Advances in cancer research 94, 29-86 (2005)). During AKT activation, the first step is the production of phosphatidylinositol 3,4,5 trisphosphate (PIP3) by PI3K. PDK1 and AKT bind the phospholipid PIP3 via their PH domains and are recruited to the plasma membrane. While RTK/PI3K mediated recruitment of AKT to the plasma membrane is a well characterized mechanism, mounting evidence indicate that AKT activation can occur in a PI3K-independent fashion (Carpten J D, et al. (2007) A transforming mutation in the pleckstrin homology domain of AKT1 in cancer. Nature 448: 439-444; Zhao J J, et al. (2006) The p110alpha isoform of PI3K is essential for proper growth factor signaling and oncogenic transformation. Proc Natl Acad Sci USA 103: 16296-16300; Sun M, et al. (2001) AKT1/PKBalpha kinase is frequently elevated in human cancers and its constitutive activation is required for oncogenic transformation in NIH3T3 cells. Am J Pathol 159: 431-437; Stemke-Hale K, et al. (2008) An integrative genomic and proteomic analysis of PIK3CA, PTEN, and AKT mutations in breast cancer. Cancer Res 68: 6084-6091; Hennessy B T, et al. (2005) Exploiting the PI3K/AKT pathway for cancer drug discovery. Nat Rev Drug Discov 4: 988-1004; Gami M S, et al. (2006) Activated AKT/PKB signaling in C. elegans uncouples temporally distinct outputs of DAF-2/insulin-like signaling. BMC Dev Biol 6: 45). About a third of the breast and prostate tumors and majority of the pancreatic tumors that exhibit AKT activation, retain normal PTEN and PI3K activity (Sun M, et al. (2001) AKT1/PKBalpha kinase is frequently elevated in human cancers and its constitutive activation is required for oncogenic transformation in NIH3T3 cells. Am J Pathol 159: 431-437; Bose S, et al. (2006) The Akt pathway in human breast cancer: a tissue-array-based analysis. Mod Pathol 19: 238-245; Panigrahi A R, et al. (2004) The role of PTEN and its signalling pathways, including AKT, in breast cancer; an assessment of relationships with other prognostic factors and with outcome. J Pathol 204: 93-100). Interestingly, normal PTEN expression was also seen in breast, ovarian and prostate tumors that exhibit activated AKT (Sun M, et al. (2001) AKT1/PKBalpha kinase is frequently elevated in human cancers and its constitutive activation is required for oncogenic transformation in NIH3T3 cells. Am J Pathol 159: 431-437). While RTKs are suggested to be involved (Zhou X, et al. (2004) Activation of the Akt/mammalian target of rapamycin/4E-BP1 pathway by ErbB2 overexpression predicts tumor progression in breast cancers. Clin Cancer Res 10: 6779-6788), the molecular mechanisms regulating RTK mediated AKT activation in cancers with normal PTEN and PI3K activity is poorly understood (Tibes R, et al. (2008) PI3K/AKT pathway activation in acute myeloid leukaemias is not associated with AKT1 pleckstrin homology domain mutation. Br J Haematol 140: 344-347). Further, PIK3CA activating mutation has recently been shown to be neither necessary nor sufficient for full AKT activation in situ (Vasudevan K M, et al. (2009) AKT-independent signaling downstream of oncogenic PIK3CA mutations in human cancer. Cancer Cell 16: 21-32). Thus, collectively these data suggest the existence of additional pathways that regulate AKT activation in response to growth factors.
Accordingly, there remains an unmet need for additional biomarkers predictive of precancerous or cancerous lesions, particularly for precancerous and cancerous lesions not utilizing PI3K/PTEN-dependent activation of AKT. Additionally, there remains an important need for additional treatment regimens and therapeutics to overcome the unresponsiveness of such precancerous and cancerous lesions. The present invention further meets these important needs, and others, as will become apparent in the teachings that follow.